K-ASSAY 

KAMIYA BIOMEDICAL COMPANY

KAMIYA BIOMEDICAL COMPANY

Cat CRP ELISA
For the quantitative determination of C-Reactive Protein (CRP)
in cat biological samples

Cat. No. KT-486

For Research Use Only.

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KAMIYA BIOMEDICAL COMPANYPRODUCT INFORMATIONCat CRP ELISA
Cat. No. KT-486
INTENDED USE
The Cat CRP ELISA is a highly sensitive two-site enzyme-linked immunoassay (ELISA) for the quantitative determinationof CRP in cat biological samples. For research use only.

INTRODUCTION
Acute phase proteins are plasma proteins which increase in concentration following infection, inflammation or trauma.
The first acute phase protein to be recognized was discovered in humans by Tillet and Frances in 1930. This C-reactive
protein (CRP) is so named because it is able to effect precipitation of somatic C-polysaccharide of Streptococcus
pneumonia. CRP is an alpha globulin with a mass of 110,000 to 140,000 daltons, and composed of five identicalsubunits, which are non-covalently assembled as a cyclic pentamer. It is synthesized in the liver and, in humans, isnormally present as a trace constituent of serum at levels less than 0.3 mg/dL. The levels in serum rise quickly following
acute tissue damage and also fall very rapidly once the stimulus is removed. It has been proposed that the function of
CRP is to aid in complement activation, influence phagocytic cell function, and augment cell mediated cytotoxicity.
Investigations over the past few years have shown that quantification of these in plasma or serum can provide valuable
information in the detection, prognosis, and monitoring of disease not only in humans, but in companion animals and farmherds as well.

PRINCIPLE
The principle of the double antibody sandwich ELISA is represented in Figure 1. In this assay the CRP present insamples reacts with the anti-CRP antibodies which have been adsorbed to the surface of polystyrene microtiter wells.
After the removal of unbound proteins by washing, anti-CRP antibodies conjugated with horseradish peroxidase (HRP),
are added. These enzyme-labeled antibodies form complexes with the previously bound CRP. Following anotherwashing step, the enzyme bound to the immunosorbent is assayed by the addition of a chromogenic substrate, 3,3’,5,5’tetramethylbenzidine (TMB). The quantity of bound enzyme varies directly with the concentration of CRP in the sample
tested; thus, the absorbance, at 450 nm, is a measure of the concentration of CRP in the test sample. The quantity ofCRP in the test sample can be interpolated from the calibration curve constructed from the calibrators, and corrected forsample dilution.

Figure 1.
Anti-CRP Antibody Bound To Solid Phase│
Calibrators and Test Samples Added│
CRP * Anti-CRP Complexes Formed│
Unbound Sample Proteins Removed│
Anti-CRP-HRP Conjugate Added│
Anti-CRP-HRP * CRP * Anti-CRP
Complexes Formed│
Unbound Anti-CRP-HRP Removed│
TMB Substrate Added│
Determine Bound Enzyme Activity

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KAMIYA BIOMEDICAL COMPANYCOMPONENTS
1. Diluent Concentrate
One bottle containing 50 mL of a 5X concentrated diluent running buffer.
2. Wash Solution Concentrate
One bottle containing 50 mL of a 20X concentrated wash solution.
3. Enzyme-Antibody Conjugate Concentrate
One vial containing 150 µL of a 100X concentrated affinity-purified anti-cat CRP antibody conjugated with HRP instabilizing buffer.
4. TMB Substrate Solution
One vial containing 12 mL of TMB and hydrogen peroxide in citric acid buffer at pH 3.3.
5. Stop Solution
One vial containing 12 mL of 0.3 M sulfuric acid. WARNING: Avoid contact with skin.
6. Microtiter Plate
Twelve removable eight-well strips in well holder frame. Wells are coated with affinity-purified anti-cat CRP.
7. Cat CRP Calibrator
One vial containing a Cat CRP Calibrator.

MATERIALS REQUIRED BUT NOT PROVIDED••••••••

Precision pipettes (2 µL to 200 µL)
Test tubes
Microplate washer/aspirator
Distilled or de-ionized H2O
Microplate reader
Assorted glassware for the preparation of reagents and buffer solutions
Timer
Vortex mixer

PRECAUTIONS
1. Read the instructions carefully before beginning the assay.
2. This kit is for research use only.
3. Great care has been taken to ensure the quality and reliability of this product. However, it is possible that in certain
cases, unusual results may be obtained due to high levels of interfering factors.
4. No additives or preservatives are necessary to maintain the integrity of the specimen. Avoid azide contamination.
5. Azide and thimerosal at concentrations higher than 0.1% inhibit the enzyme reaction.
6. Other precautions: Do not interchange kit components from different lots.
 Do not use kit components beyond the expiration date.
 Protect reagents from direct sunlight.
 Do not pipette by mouth.
 Do not eat, drink, smoke or apply cosmetics where reagents are used.
 Avoid all contact with the reagents by using gloves.
 Stop solution contains diluted sulfuric acid. Irritation to eyes and skin is possible. Flush with water after contact.

REAGENT PREPARATION
1. Diluent Concentrate
The Diluent solution supplied is a 5X concentrate and must be diluted 1:5 with distilled or de-ionized water.
2. Wash Solution Concentrate
The Wash Solution supplied is a 20X concentrate and must be diluted 1:20 with distilled or de-ionized water. Crystal
formation in the concentrate is not uncommon when storage temperatures are low. Warming of the concentrate to30-35°C before dilution can dissolve crystals.
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3. Enzyme-Antibody Conjugate Concentrate
Calculate the required amount of working conjugate solution for each microtiter plate test strip by adding 10 µL
Enzyme-Antibody Conjugate to 990 µL of 1X Diluent for each test strip to be used for testing. Mix uniformly, but
gently. Avoid foaming.
4. TMB Substrate Solution
Ready to use as supplied.
5. Stop Solution
Ready to use as supplied.
6. Microtiter Plate
Ready to use as supplied. Unseal microtiter pouch and remove plate from pouch. Remove all strips and wells that will
not be used in the assay and place back in pouch and re-seal along with desiccant.
7. Cat CRP Calibrator
The Cat CRP Calibrator should be aliquoted out and stored frozen. The calibrator is now at a concentration of 88.4µg/mL and needs to be diluted in 1X diluent according to the chart below for each run. Cat CRP Calibrators need tobe prepared immediately prior to use (see chart below). Mix well between each step. Avoid foaming.
Calibrator
A
765
4
3
2
10

Concentration(ng/mL)
884
200
100
5025
12.5
6.25
3.125
0Calibrator Volumeadded to 1X Diluent
5 µL Cat CRP Calibrator
150 µL Calibrator A
300 µL Calibrator 7300 µL Calibrator 6300 µL Calibrator 5300 µL Calibrator 4300 µL Calibrator 3300 µL Calibrator 2

Volume of 1X Diluent
495 µL513 µL
300 µL
300 µL
300 µL
300 µL
300 µL
300 µL500 µLSTORAGE AND STABILITY
1. Complete Kit
The expiration date for the kit is stated on the outer label. The recommended storage temperature is 4°C. Note: Seelong term storage recommendations below for the Cat CRP Calibrator.
2. Diluent
The 5X Diluent Concentrate is stable until the expiration date. The 1X working solution is stable for at least one weekfrom the date of preparation. Both solutions should be stored at 4°C.
3. Wash Solution
The 20X Wash Solution Concentrate is stable until the expiration date. The 1X working solution is stable for at least
one week from the date of preparation. Both solutions can be stored at room temperature (RT, 16-25°C) or at 4°C.
4. Enzyme-Antibody Conjugate
Undiluted anti-CRP-HRP conjugate should be stored at 4°C and diluted immediately prior to use. The workingconjugate solution is stable for up to 8 hours.
5. TMB Substrate Solution
The TMB Substrate Solution should be stored at 4°C and is stable until the expiration date.
6. Stop Solution
The Stop Solution should be stored at 4°C and is stable until the expiration date.

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7. Microtiter Plate
Anti-cat CRP coated wells are stable until the expiration date, and should be stored at 4°C in the sealed foil pouchwith desiccant pack.
8. Cat CRP Calibrator
Long Term Storage: Upon receipt, aliquot the calibrator and store them frozen. They will be stable until the expiration
date. Short Term Storage: The calibrator is stable for up to 14 days at 4°C. The working calibrator solutions should beprepared immediately prior to use and are stable for up to 8 hours.

INDICATIONS OF INSTABILITY
If the test is performing correctly, the results observed with the calibrator solutions should be within 20% of the expectedvalues.

SPECIMEN COLLECTION AND HANDLING
Blood should be collected by venipuncture and the serum separated from the cells, after clot formation, by centrifugation.
For plasma samples, blood should be collected into a container with an anticoagulant and then centrifuged. Care should
be taken to minimize hemolysis, excessive hemolysis can impact your results. Assay immediately or aliquot and store
samples at -20°C. Avoid repeated freezing/thawing.
For any sample that might contain pathogens, care must be taken to prevent contact with open wounds. No additives orpreservatives are necessary to maintain the integrity of the specimen. Avoid azide contamination.

ASSAY PROTOCOL
Dilution of Samples
Due to the high sensitive nature of the assay each sample should be diluted before use for a normal assay. A 1:8,000
dilution of serum or plasma is appropriate for most samples. For absolute quantification of samples that yield results
outside the range of the calibration curve, a lesser or greater dilution might be required. If unsure of sample level, a serial
dilution with one or two representative samples before running the entire plate is highly recommended.
To prepare a 1:8,000 dilution of sample, transfer 5 µL of sample to 495 µL of 1X Diluent. This gives you a 1:100 dilution.
Mix thoroughly. Next, dilute the 1:100 samples by transferring 5 µL to 395 µL of 1X Diluent. You now have a 1:8,000
dilution of your sample. Mix thoroughly at each stage.

Procedure
Bring all reagents to RT before use.

1. Pipette 100 µL of
Calibrator 0 (0.0 ng/mL) in duplicate
Calibrator 1 (3.125 ng/mL) in duplicate
Calibrator 2 (6.25 ng/mL) in duplicate
Calibrator 3 (12.5 ng/mL) in duplicate
Calibrator 4 (25 ng/mL) in duplicate
Calibrator 5 (50 ng/mL) in duplicate
Calibrator 6 (100 ng/mL) in duplicate
Calibrator 7 (200 ng/mL) in duplicate
2. Pipette 100 µL of diluted sample (in duplicate) into pre designated wells.
3. Incubate the Microtiter Plate at 22°C (RT) for thirty (30 ± 2) minutes. Keep plate covered and level during incubation.
4. Following incubation, aspirate the contents of the wells.
5. Completely fill each well with appropriately diluted Wash Solution and aspirate. Repeat three times, for a total of fourwashes. If washing manually: completely fill wells with diluted Wash Solution, invert the plate and pour/shake out the5Rev. 032009

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contents in a waste container. Follow this by sharply striking the wells on absorbent paper to remove residual Wash
Solution. Repeat three times for a total of four washes.
6. Pipette 100 µL of appropriately diluted Enzyme-Antibody Conjugate to each well. Incubate at 22°C (RT) for thirty (30± 2) minutes. Keep plate covered in the dark and level during incubation.
7. Wash and blot the wells as described in Steps 4 and 5.
8. Pipette 100 µL of TMB Substrate Solution into each well.
9. Incubate in the dark at RT for precisely ten (10) minutes.
10. After ten minutes, add 100 µL of Stop Solution to each well.
11. Determine the absorbance at 450 nm of the contents of each well. Zero the plate reader to air.
The absorbance of the final reaction mixture can be measured up to 2 hours after the addition of the Stop Solution.
However, good laboratory practice dictates that the measurement be made as soon as possible.

RESULTS
1. Subtract the average background value from the test values for each sample.
2. Using the results observed for the calibrators construct a calibration curve. The appropriate curve fit is that of a fourparameter logistics curve, although a second order polynomial (quadratic) or other curve fits may also be used.
3. Interpolate test sample values from calibration curve. Correct for sample dilution factor to arrive at CRP concentrationin original sample.

PERFORMANCE CHARACTERISTICS
In accord with good laboratory practice, the assays for specific CRP require meticulous quality control. Each laboratory
should use routine quality control procedures to establish inter- and intra-assay precision and performance characteristics.

LIMITATION OF THE PROCEDURE
1. Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete
understanding of the information contained in the package insert instructions and with adherence to good laboratory
practice.
2. Factors that might affect the performance of the assay include proper instrument function, cleanliness of glassware,
quality of distilled or de-ionized water, and accuracy of reagent and sample pipettings, washing technique, incubation
time or temperature.

FOR RESEARCH USE ONLY

KAMIYA BIOMEDICAL COMPANY
12779 Gateway Drive, Seattle, WA 98168
Tel: (206) 575-8068 Fax: (206) 575-8094
Email: LifeScience@k-assay.com
www.k-assay.com

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